| Aptamers are short single-stranded oligonucleotide sequences with high affinity and specificity for certain targets screened through the Systematic Evolution of Ligands by Exponential Enrichment(SELEX) technique from the random oligonucleotide library. Capillary Electrophoresis(CE) is a powerful separation technology with high efficiency, fast analysis speed, low cost, various separation modes. Inherited the advantages of CE, CE-SELEX technology can quickly separate target-nucleic acidcompounds, improving the efficiencyof the SELEX. In this thesis, a CE-SELEX method screening for aptamers against small molecule targets was established, and single-stranded DNA aptamers against clenbuterol was selected. There are mainly five parts in this thesis.(1) The concept of aptamer, the principle of SELEX, research advances of aptamers selection for small molecules targets were reviewed.The characteristics and analysis models of CE, and the advantages of CE-SELEX were summerized. The content and significance of this thesis were described.(2) A CE analysis method for clenbuterol was established, and the interaction of clenbuterol with random oligonucleotide library were characterized by CE.(3) The PCR process in SELEX was characterized by CE and Fluorescence Real-Time Quantitative PCR(Q-PCR), and then the annealing temperature and cycle number of PCR process, the primers ration of asymmetry PCR for the preparation of single-stranded secondary library was optimized.(4) 80 mer random oligonucleotide library was mixed with targets, the binding DNA molecules were collected and amplified, prepared into the single-standed secondary library by asymmetric PCR. Four rounds of CE-SELEX were conducted, the affinity increase of each round was characterized.(5) After 4 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 13 sequences were simulated for their secondary structures by the DNAMAN software. The dissociation constant of aptamer c12 was determinated. |
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